EP4110802A1 - Thérapie basée sur une protéine de fusion fc du domaine extracellulaire cd80 - Google Patents

Thérapie basée sur une protéine de fusion fc du domaine extracellulaire cd80

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Publication number
EP4110802A1
EP4110802A1 EP21713285.1A EP21713285A EP4110802A1 EP 4110802 A1 EP4110802 A1 EP 4110802A1 EP 21713285 A EP21713285 A EP 21713285A EP 4110802 A1 EP4110802 A1 EP 4110802A1
Authority
EP
European Patent Office
Prior art keywords
fusion protein
administered
ecd
seq
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21713285.1A
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German (de)
English (en)
Inventor
Siddhartha Mitra
Maike Schmidt
Kathleen M. Sullivan
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Five Prime Therapeutics Inc
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Five Prime Therapeutics Inc
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Filing date
Publication date
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Publication of EP4110802A1 publication Critical patent/EP4110802A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • FIELD FIELD
  • This present disclosure relates to methods of administering fusion proteins comprising an CD80 (B7-1) extracellular domain (ECD) and an immunoglobulin fragment crystallizable (Fc) domain, optionally, in combination with a PD-1/PD-L1 antagonist, such as pembrolizumab, for the treatment of diseases such as cancer.
  • Advantageous dose regimens are provided.
  • T-cell regulation involves the integration of multiple signaling pathways: signaling via the T-cell receptor (TCR) complex and through co-signaling receptors, both co- stimulatory and co-inhibitory.
  • CD80 cluster of differentiation 80, also known as B7, B7.1, B7-1) is a well-characterized co-signaling ligand.
  • CD80 acts as a co-stimulatory ligand via interactions with its receptor, cluster of differentiation 28 (CD28), expressed on T-cells.
  • CD80 also interacts with co-inhibitory molecules cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-ligand 1 (PD-L1).
  • CTLA-4 cytotoxic T-lymphocyte-associated antigen-4
  • PD-L1 programmed death-ligand 1
  • the co-stimulatory and co-inhibitory ligands ensure both tolerance to self-antigens and the ability to mount an appropriate immune response to non-self antigens.
  • the immune system is often initially able to mount an effective immune response against tumor cells via TCR-dependent and -independent mechanisms, some tumors can evade the immune response. Mechanisms by which this occurs include the upregulation of pathways that enforce peripheral tolerance to self-antigens (including CTLA-4 and PD-L1).
  • Recent immuno-oncology approaches have focused on reprogramming the immune system to mount an effective immune response against tumors that have evaded the initial immune response.
  • checkpoint inhibitors For example, blocking antibodies against both the programmed cell death protein (PD-1)/PD-L1 and CTLA-4 axes have been effective in anti-tumor immunity, including improved progression free survival (PFS) and overall survival (OS) in some patients.
  • PFS progression free survival
  • OS overall survival
  • responses have only been observed in select tumor types, within which only a fraction of patients respond to checkpoint inhibitors.
  • some patients do achieve long term disease control with the use of blocking antibodies against the PD-1/PD-L1 and CTLA-4 axes, the majority of patients either do not respond or respond then subsequently relapse.
  • PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression.
  • PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA.
  • PD-1 Programmed Death Ligand-1
  • PD-L2 Programmed Death Ligand-2
  • PD-L1 Programmed Death Ligand-1
  • PD-L2 Programmed Death Ligand-2
  • Inhibition of the PD-1/PD-L1 interaction e.g., by anti- PD-1 or anti-PD-L1 antibodies, mediates antitumor activity.
  • the combination of nivolumab, an anti-PD-1 antibody, and ipilimumab, an anti-CTLA4 antibody has demonstrated superior efficacy in advanced melanoma compared to either agent alone.
  • nivolumab resulted in an objective response rate (ORR) of 25% and median progression-free survival (PFS) of 4.6 months.
  • ORR objective response rate
  • PFS median progression-free survival
  • the combination of ipilimumab and nivolumab resulted in an ORR of 42% and median PFS of 11.6 months.
  • ORR objective response rate
  • PFS median progression-free survival
  • a fusion protein comprising the extracellular domain (ECD) of human cluster of differentiation 80 (CD80) and the fragment crystallizable (Fc) domain of human immunoglobulin G 1 (IgG1).
  • ECD extracellular domain
  • Fc fragment crystallizable domain of human immunoglobulin G 1
  • the fusion protein can be administered to treat a solid tumor in a subject. In some aspects, about 0.07 mg to 700 mg of the fusion protein is administered.
  • the fusion protein can be administered once every three weeks, once every two weeks, or once a week.
  • the fusion can be administered in combination with a PD-1/PD-L1 antagonist (e.g., pembrolizumab).
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 0.07 mg to about 700 mg of a fusion protein comprising the extracellular domain (ECD) of human cluster of differentiation 80 (CD80) and the fragment crystallizable (Fc) domain of human immunoglobulin G 1 (IgG1) and (ii) a PD-1/PD-L1 antagonist.
  • ECD extracellular domain
  • CD80 human cluster of differentiation 80
  • Fc fragment crystallizable domain of human immunoglobulin G 1
  • a PD-1/PD-L1 antagonist a PD-1/PD-L1 antagonist
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 0.07 mg to about 700 mg of a fusion protein comprising the ECD of CD80 and the Fc domain of human IgG1 and (ii) about 200 mg of an anti-PD-1 antibody or antigen-binding fragment thereof comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:16, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:17.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 0.07 mg to about 700 mg of a fusion protein comprising the ECD of CD80 and the Fc domain of human IgG1, wherein the fusion protein is administered once every two weeks or once every week.
  • about 21 mg to about 700 mg of the fusion protein is administered.
  • about 70 mg to about 700 mg of the fusion protein is administered.
  • about 280 mg of the fusion protein is administered.
  • about 210 mg of the fusion protein is administered.
  • about 140 mg of the fusion protein is administered.
  • about 70 mg of the fusion protein is administered.
  • about 42 mg of the fusion protein is administered. In some aspects, about 21 mg of the fusion protein is administered. In some aspects, about 700 mg of the fusion protein is administered. In some aspects, about 630 mg of the fusion protein is administered. In some aspects, about 560 mg of the fusion protein is administered. In some aspects, about 420 mg of the fusion protein is administered. In some aspects, about 7 mg of the fusion protein is administered. In some aspects, about 2.1 mg of the fusion protein is administered. In some aspects, about 0.7 mg of the fusion protein is administered. In some aspects, about 0.21 mg of the fusion protein is administered. In some aspects, about 0.07 mg of the fusion protein is administered. [0013] In some aspects, the fusion protein is administered once every three weeks.
  • the fusion protein is administered once every two weeks. In some aspects, the fusion protein is administered once a week. [0014] In some aspects, the fusion protein is administered intravenously. [0015] In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof is a PD-1 antagonist. [0016] In some aspects, the PD-1/PD-L1 antagonist is an anti-PD-1 antibody or antigen- binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, or a soluble polypeptide. [0017] In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof is administered once every three weeks.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered intravenously.
  • the fusion protein and the anti-PD-1 antibody or antigen-binding fragment thereof are administered as separate formulations on the same day.
  • the fusion protein and the anti-PD-1 antibody or antigen-binding fragment thereof are administered sequentially.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered after the fusion protein is administered.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered about 15 minutes to about 3 hours after the fusion protein is administered.
  • the fusion protein and the anti-PD-1 antibody or antigen-binding fragment thereof are administered concurrently.
  • the ECD of human CD80 comprises the amino acid sequence set forth in SEQ ID NO:1.
  • the Fc domain of human IgG1 comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the Fc domain of human IgG1 is linked to the carboxy terminus of the ECD of human CD80.
  • the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:5.
  • the fusion protein comprises at least 20 molecules of SA. In some aspects, the fusion protein comprises at least 15 molecules of SA.
  • the fusion protein comprises 15-60 molecules of SA. In some aspects, the fusion protein comprises 15-40 molecules of SA. In some aspects, the fusion protein comprises 15-30 molecules of SA. In some aspects, the fusion protein comprises 20-30 molecules of SA. [0024] In some aspects, the fusion protein is administered in a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition comprises at least 20 moles of SA per mole of fusion protein. In some aspects, the pharmaceutical composition comprises at least 15 moles of SA per mole of fusion protein. In some aspects, the pharmaceutical composition comprises 15- 60 moles of SA per mole of fusion protein. In some aspects, the pharmaceutical composition comprises 15-40 moles of SA per mole of fusion protein.
  • the pharmaceutical composition comprises 15-30 moles of SA per mole of fusion protein. In some aspects, the pharmaceutical composition comprises 20-30 moles of SA per mole of fusion protein.
  • the anti-PD-1 antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO:10 and a VL comprising the amino acid sequence SEQ ID NO:11. In some aspects, the anti-PD-1 antibody or antigen- binding fragment is pembrolizumab.
  • the solid tumor is an advanced solid tumor. In some aspects, the solid tumor is not a primary central nervous system tumor.
  • the solid tumor is a colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, endometrial cancer, or sarcoma.
  • the solid tumor is lung cancer.
  • the solid tumor is non-small cell lung cancer.
  • the patient has not received prior therapy with a PD-1/PD-L1 antagonist.
  • the patient has received prior therapy with at least one PD-1/PD-L1 antagonist selected from a PD-L1 antagonist and a PD-1 antagonist.
  • the at least one PD-1/PD-L1 antagonist is nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. In some aspects, the at least one PD-1/PD-L1 antagonist was administered in an advanced or metastatic setting.
  • the patient has received prior therapy with at least one anti- angiogenic agent.
  • the anti-angiogenic agent is sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. In some aspects, the anti- angiogenic agent was administered in an advanced or metastatic setting.
  • the patient has a BRAF mutation.
  • the patient has received prior therapy with at least one BRAF inhibitor.
  • the BRAF inhibitor is vemurafenib or dabrafenib.
  • the BRAF inhibitor was administered in an advanced or metastatic setting.
  • the solid tumor is recurrent or progressive after a therapy selected from surgery, chemotherapy, radiation therapy, and a combination thereof BRIEF DESCRIPTION OF THE DRAWINGS
  • Figs.1a-d show release of cytokines IFN- ⁇ and TNF- ⁇ from T-cells on 96-well tissue culture plates exposed to protein A beads coated with 0.01, 0.1, or 1 ⁇ g/well of a CD80 ECD IgG1 Fc domain fusion molecule (CD80-Fc).
  • Figs.1a and 1c show that bead- immobilized CD80-Fc alone did not cause significant T-cell activation, as measured by soluble cytokine production.
  • Figs.1b and 1d show that when a small amount of OKT3- scFv (too low to cause T-cell stimulation on its own) was immobilized along with the CD80-Fc, cytokine release was observed.
  • Fig.2 shows tumor growth of murine CT26 tumors following treatment with a saline control or either 0.3 or 0.6 mg/kg doses of three different lots of a CD80 ECD-Fc fusion molecule having three different sialic acid (SA) contents.
  • SA sialic acid
  • Treatment with CD80 ECD-Fc lot A at 0.3 mg/kg did not inhibit tumor growth compared to the control and when dosed at 0.6 mg/kg it only induced 70% inhibition (P ⁇ 0.001) of tumor growth.
  • Fig.3 shows tumor growth of CT26 tumors treated with mouse IgG2b at 10 mg/kg; murine CD80 ECD-Fc SA 20 mol/mol at 0.3 mg/kg; anti-CTLA4 antibody clone 9D9 at 10 mg/kg; and anti-CTLA4 antibody clone 9D9 at 1.5 mg/kg.
  • Arrows indicate when mice were dosed.
  • the asterisk symbol (*) denotes statistically significant differences between murine CD80 ECD-Fc SA 20 mol/mol at 0.3 mg/kg and the other treatments.
  • Fig.4 shows tumor growth of MC38 tumors treated with mouse IgG2b at 10 mg/kg; murine CD80 ECD-Fc SA 20 mol/mol at 3 mg/kg; anti-CTLA4 antibody clone 9D9 at 10 mg/kg; and anti-CTLA4 antibody clone 9D9 at 1.5 mg/kg.
  • Arrows indicate when mice were dosed.
  • the asterisk symbol (*) denotes statistically significant differences between murine CD80 ECD-Fc SA 20 mol/mol at 3 mg/kg and the other treatments.
  • Fig.7 shows normalized expression of granzyme B (Gzmb) and interferon gamma (Ifng) in the tumor cells and in the blood of BALB/c mice inoculated with CT26 colorectal carcinoma cells and in the blood of na ⁇ ve BALB/c mice.
  • the CT26 tumor- bearing mice and the na ⁇ ve mice received either mIgG2a (control) or a dose of murine CD80 ECD-Fc.
  • the asterisk symbol (*p ⁇ 0.05) or (**p ⁇ 0.01) denotes statistically significant differences between murine CD80 ECD-Fc compared to control treatment. (See Example 11).
  • Figs.8a and b show hCD80ECD:hIgG1Fc-induced stimulator-dependent allogeneic T cell cytokine secretion.
  • Whole blood was added to two amounts of pooled, irradiated PBMC and cultured for 5 days following the addition of multiple doses of Fc-Hinge control or hCD80ECD:hIgG1Fc. All data are mean ⁇ SD of the mean of 6 technical replicates from 6 individual donors.
  • Figs.9a and b show hCD80ECD:hIgG1Fc-induced stimulator-dependent T cell costimulation.
  • (9a) Increased proliferation of CD4 and CD8 T cells stimulated with hCD80ECD:hIgG1Fc as determined by EdU incorporation.
  • Fig.10 shows the impact of murine CD80 ECD-Fc on the growth of CT26 tumors.
  • the average tumor growth (left graph) and individual tumor volumes of all groups on day 21 (right graph) are shown.
  • Immunocompetent BALB/c mice were inoculated with 1x10 6 CT26 tumor cells.
  • Treatment with murine CD80 ECD-Fc was initiated on day 10; three doses were administered on days 10, 13, and 17.
  • Murine CD80 ECD-Fc significantly inhibited tumor growth (**** indicates p ⁇ 0.0001 for 0.3 mg/kg; ** indicates p ⁇ 0.01 for 1 mg/kg, and *** p ⁇ 0.001 for 3 mg/kg).
  • Statistical significance was determined by 1-way ANOVA.
  • Fig.11 shows the serum concentration versus time profiles of hCD80ECD:hIgG1Fc in patients who received a single intravenous infusion administration of hCD80ECD:hIgG1Fc in the dose range of 0.07 mg to 42 mg.
  • Figs.12a and b show the activity of murine CD80 ECD-Fc and an anti-PD1 antibody against CT26 tumors.
  • CTRs complete tumor regressions.
  • polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a "polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity.
  • a “fusion molecule” as used herein refers to a molecule composed of two or more different molecules that do not occur together in nature being covalently or noncovalently joined to form a new molecule.
  • fusion molecules may be comprised of a polypeptide and a polymer such as PEG, or of two different polypeptides.
  • a “fusion protein” refers to a fusion molecule composed of two or more polypeptides that do not occur in a single molecule in nature.
  • a “CD80 extracellular domain” or “CD80 ECD” refers to an extracellular domain polypeptide of CD80, including natural and engineered variants thereof.
  • a CD80 ECD can, for example, comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:1 or 2.
  • a “CD80 ECD fusion molecule” refers to a molecule comprising a CD80 ECD and a fusion partner. The fusion partner may be covalently attached, for example, to the N- or C- terminal of the CD80 ECD or at an internal location.
  • a “CD80 ECD fusion protein” is a CD80 ECD fusion molecule comprising a CD80 ECD and another polypeptide that is not naturally associated with the CD80 ECD, such as an Fc domain.
  • a CD80 ECD fusion protein can, for example, comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 4 or 5.
  • the term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • an antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity.
  • An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
  • the term “antibody fragment” refers to a portion of an intact antibody.
  • An “antigen-binding fragment,” “antigen-binding domain,” or “antigen-binding region,” refers to a portion of an antibody that binds to an antigen.
  • An antigen-binding fragment can contain an antigen recognition site of an intact antibody (e.g., complementarity determining regions (CDRs) sufficient to specifically bind antigen).
  • CDRs complementarity determining regions
  • antigen-binding fragments of antibodies include, but are not limited to Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, and single chain antibodies.
  • An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.
  • the terms “anti-PD-1 antibody,” “PD-1 antibody” and “antibody that binds to PD-1” refer to an antibody that is capable of specifically binding PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-1.
  • anti-PD-L1 antibody refers to an antibody that is capable of specifically binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-L1.
  • specifically binding refers to an antibody that is capable of specifically binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-L1.
  • specifically binding is analogous terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen- binding domain and that the binding entails some complementarity between the antigen- binding domain and the epitope.
  • an antibody that “specifically binds” to human PD-1 may also bind to PD-1 from other species (e.g., cynomolgus monkey, mouse, and/or rat PD-1) and/or PD-1 proteins produced from other human alleles, but the extent of binding to an un-related, non-PD-1 protein is less than about 10% of the binding of the antibody to PD-1 as measured, e.g., by a radioimmunoassay (RIA).
  • a “monoclonal” antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in the highly specific binding of a single antigenic determinant, or epitope.
  • monoclonal antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site.
  • monoclonal antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • the variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • variable region is a human variable region.
  • variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • FRs human framework regions
  • the variable region is a primate (e.g., non-human primate) variable region.
  • the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).
  • VH and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.
  • VL and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.
  • Kabat numbering and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof.
  • CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242).
  • CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3).
  • CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).
  • the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.
  • Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.
  • the term “constant region” and “constant domain” are interchangeable and have their common meanings in the art.
  • the constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
  • the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • an antibody or antigen- binding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ), and mu ( ⁇ ), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG 1 , IgG 2 , IgG 3 , and IgG 4 .
  • Heavy chain amino acid sequences are well known in the art.
  • the heavy chain is a human heavy chain.
  • the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa ( ⁇ ) or lambda ( ⁇ ) based on the amino acid sequence of the constant domains.
  • Light chain amino acid sequences are well known in the art.
  • the light chain is a human light chain.
  • isolated refers to a molecule that has been separated from at least some of the components with which it is typically found in nature.
  • a polypeptide or antibody is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced.
  • a polypeptide or antibody is secreted by a cell after expression, physically separating the supernatant containing the polypeptide or antibody from the cell that produced it is considered to be “isolating” the polypeptide or antibody.
  • a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide.
  • a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated” so long as that polynucleotide is not found in that vector in nature.
  • subject and “patient” are used interchangeably herein to refer to a human.
  • methods of treating other mammals including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided.
  • cancer is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth.
  • a cancer can be a solid tumor, for example, a colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, endometrial cancer, or sarcoma.
  • Terms such as “treating,” “treatment,” and “to treat,” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a pathologic condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder.
  • a subject is successfully “treated” for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non- tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof.
  • PFS progression-free survival
  • DFS disease-
  • administer refers to methods that may be used to enable delivery of a drug, e.g., a CD80 ECD fusion protein to the desired site of biological action (e.g., intravenous administration).
  • Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.
  • the term “therapeutically effective amount” refers to an amount of a drug, e.g., a CD80 ECD fusion protein, effective to treat a disease or disorder in a subject.
  • the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size or burden; inhibit, to some extent, cancer cell infiltration into peripheral organs; inhibit, to some extent, tumor metastasis; inhibit, to some extent, tumor growth; relieve, to some extent, one or more of the symptoms associated with the cancer; and/or result in a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP), or any combination thereof.
  • PFS progression-free survival
  • DFS disease-free survival
  • OS overall survival
  • CR complete response
  • PR partial response
  • SD stable disease
  • SD stable disease
  • PD progressive disease
  • resistant when used in the context of treatment with a therapeutic agent, means that the subject shows decreased response or lack of response to a standard dose of the therapeutic agent, relative to the subject’s response to the standard dose of the therapeutic agent in the past, or relative to the expected response of a similar subject with a similar disorder to the standard dose of the therapeutic agent.
  • a subject may be resistant to a therapeutic agent although the subject has not previously been given the therapeutic agent, or the subject may develop resistance to the therapeutic agent after having responded to the agent on one or more previous occasions.
  • a “refractory” cancer is one that progresses even though an anti-tumor treatment, such as a chemotherapy, is administered to the cancer patient.
  • a “recurrent” cancer is one that has regrown, either at the initial site or at a distant site, after a response to initial therapy.
  • the terms “programmed cell death protein 1” and “PD-1” refer to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T-cells in vivo, and binds to two ligands, PD-L1 and PD-L2.
  • the term "PD-1" as used herein includes human PD-1 (hPD-1), naturally occurring variants and isoforms of hPD-1, and species homologs of hPD-1. A mature hPD-1 sequence is provided as SEQ ID NO:6.
  • the terms “programmed cell death 1 ligand 1” and “PD-L1” refer to one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that down regulate T-cell activation and cytokine secretion upon binding to PD-1.
  • the term "PD-L1" as used herein includes human PD-L1 (hPD-L1), naturally occurring variants and isoforms of hPD-1, and species homologs of hPD-L1. A mature hPD-L1 sequence is provided as SEQ ID NO:7.
  • the term “PD-1/PD-L1 antagonist” refers to a moiety that disrupts the PD-1/PD-L1 signaling pathway.
  • the antagonist inhibits the PD-1/PD-L1 signaling pathway by binding to PD-1 and/or PD-L1. In some aspects, the PD-1/PD-L1 antagonist also binds to PD-L2. In some aspects, a PD-1/PD-L1 antagonist blocks binding of PD-1 to PD-L1 and optionally PD-L2.
  • Nonlimiting exemplary PD-1/PD-L1 antagonists include PD-1 antagonists, such as antibodies that bind to PD-1 (e.g., nivolumab and pembrolizumab); PD-L1 antagonists, such as antibodies that bind to PD-L1 (e.g., atezolizumab, durvalumab, and avelumab); fusion proteins, such as AMP-224; and peptides, such as AUR-012.
  • PD-1 antagonists such as antibodies that bind to PD-1 (e.g., nivolumab and pembrolizumab); PD-L1 antagonists, such as antibodies that bind to PD-L1 (e.g., atezolizumab, durvalumab, and avelumab); fusion proteins, such as AMP-224; and peptides, such as AUR-012.
  • PD-1 antagonists such as antibodies that bind to PD-1 (e.g.,
  • an “anti-angiogenic agent” or “angiogenesis inhibitor” refers to an agent such as a small molecular weight substance, a polynucleotide (including, e.g., an inhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly.
  • RNAi or siRNA inhibitory RNA
  • an anti-angiogenic agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor.
  • an anti-angiogenic agent is an antibody to or other antagonist of an angiogenic agent, e.g., antibodies to VEGF-A (e.g., bevacizumab (Avastin ® )) or to the VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec ® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, Sutent ® /SU11248 (sunitinib malate), AMG706, or those described in, e.g., international patent application WO 2004/113304).
  • VEGF-A e.g., bevacizumab (Avastin ® )
  • VEGF-A receptor e.g., KDR receptor or Flt-1 receptor
  • anti-PDGFR inhibitors such as Gleevec ® (imat
  • Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’Amore (1991) Annu. Rev. Physiol.53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2 listing known anti-angiogenic factors); Sato (2003) Int. J. Clin.
  • native angiogenesis inhibitors e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’Amore (1991) Annu. Rev. Physiol.53:217-39; Streit and De
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • the formulation can be sterile.
  • a pharmaceutical composition may contain a “pharmaceutical carrier,” which refers to carrier that is non- toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • the carrier ideally is not irritable to the skin and does not cause injection site reaction.
  • active agents e.g., CD80 ECD-Fc fusion protein and a PD-1/PD-L1 antagonist (e.g., pembrolizumab)
  • a PD-1/PD-L1 antagonist e.g., pembrolizumab
  • administration of active agents can provide “synergy” or be “synergistic, ” i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects that results from using the active ingredients separately.
  • a synergistic effect can be attained when the active ingredients are: (1) co- formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered serially, by alternation, or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes.
  • CD80 Extracellular Domain Fc Fusion Proteins Provided herein are methods of administering CD80 ECD fusion proteins comprising a CD80 ECD and an Fc domain (a “CD80 ECD-Fc fusion protein”). The fusion proteins can be administered in combination with a PD-1/PD-L1 antagonist.
  • the CD80 ECD can, for example, be a human CD80 ECD.
  • the human CD80 ECD comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:1.
  • the Fc domain can be the Fc domain of an IgG.
  • the Fc domain can be the Fc domain of a human immunoglobulin.
  • the Fc domain is a human IgG Fc domain.
  • the Fc domain is a human IgG1 Fc domain.
  • the human IgG1 Fc domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:4.
  • the CD80 ECD and the Fc domain can be directly linked such that the N-terminal amino acid of the Fc domain immediately follows the C-terminal amino acid of the CD80 ECD.
  • the CD80 ECD and the Fc domain are translated as a single polypeptide from a coding sequence that encodes both the CD80 ECD and the Fc domain.
  • the Fc domain is directly fused to the carboxy-terminus of the CD80 ECD polypeptide.
  • the CD80 ECD-Fc fusion protein comprises a human CD80 ECD and a human IgG1 Fc domain.
  • the CD80 ECD-Fc fusion protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:5.
  • CD80 ECD-Fc fusion proteins can, depending on how they are produced, have different levels of particular glycosylation modifications.
  • a CD80 ECD-Fc fusion protein can have different amounts of sialic acid (SA) residues.
  • SA sialic acid
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 10 to 60 molecules of SA.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 15 to 60 molecules of SA.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 10 to 40 molecules of SA. In certain aspects, a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 15 to 30 molecules of SA. In certain aspects, a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 15 to 25 molecules of SA. In certain aspects, a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 20 to 40 molecules of SA. In certain aspects, a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 20 to 30 molecules of SA.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 30 to 40 molecules of SA.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises 10, 15, 20, 25, 30, 35, or 40 molecules of SA.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises at least 15 molecules of SA.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises at least 20 molecules of SA.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises at least 25 molecules of SA. In certain aspects, a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises at least 30 molecules of SA. In certain aspects, a CD80 ECD- Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises at least 35 molecules of SA. In certain aspects, a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) comprises at least 40 molecules of SA. [0087] CD80 ECD-Fc fusion proteins can directly engage CD28 through the CD80 ECD.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) is capable of activating na ⁇ ve and memory T cells.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) is capable of directly activating na ⁇ ve and memory T cells.
  • CD80 ECD-Fc fusion proteins can also bind to CTLA-4 through the CD80 ECD. This can cause de-repressing T-cell activation by enabling the interaction of endogenous CD20 with CD28 at the immune synapse.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) is capable of binding to CD28.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) is capable of binding to CTLA-4.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) is capable of binding to CD28 and CTLA-4.
  • a CD80 ECD-Fc fusion protein is not a CD28 superagonist.
  • a CD80 ECD-Fc fusion protein (e.g., comprising SEQ ID NO:5) is at least 1000-fold less potent at inducing cytokine release compared to TGN1412. 3.
  • PD-1/PD-L1 Antagonists [0091] Provided herein are methods of administering a PD-1/PD-L1 antagonist in combination with a CD80 ECD fusion proteins comprising a CD80 ECD and an Fc domain (a “CD80 ECD-Fc fusion protein”).
  • the PD-1/PD-L1 antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, or a soluble polypeptide (e.g., AMP-224 orAUR-012).
  • the PD-1 antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof.
  • an anti-PD-1 antibody or antigen-binding fragment thereof is pembrolizumab or an antigen-binding fragment thereof or nivolumab or an antigen-binding fragment thereof.
  • the PD-1 antagonist is pembrolizumab.
  • the heavy and light chain sequences of pembrolizumab are listed in the following table. In the context of the heavy and light chain sequences, CDR sequences are shown in bold, and the variable region sequences are underlined.
  • the PD-1 antagonist is antibody or antigen-binding fragment comprising the heavy and light chain variable region CDRs of pembrolizumab (e.g., the CDR sequences provided in the table above, the Kabat-defined CDRs, the AbM-defined CDRs, or the Chothia-defined CDRs).
  • the PD-1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of pembrolizumab.
  • the PD-1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable region CDRs of nivolumab (e.g., the Kabat- defined CDRs, the AbM-defined CDRs, or the Chothia-defined CDRs).
  • the PD-1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of nivolumab.
  • the PD-L1 antagonist is an anti-PD-L1 antibody or antigen-binding fragment thereof.
  • an anti-PD-L1 antibody or antigen-binding fragment thereof is atezolizumab or an antigen-binding fragment thereof, durvalumab or an antigen-binding fragment thereof, or avelumab or an antigen-binding fragment thereof.
  • the PD-L1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable region CDRs of atezolizumab (e.g., the Kabat-defined CDRs, the AbM-defined CDRs, or the Chothia-defined CDRs).
  • the PD-L1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of atezolizumab.
  • the PD-L1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable region CDRs of durvalumab (e.g., the Kabat-defined CDRs, the AbM-defined CDRs, or the Chothia-defined CDRs). In one aspect, the PD-L1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of durvalumab.
  • the PD-L1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable region CDRs of avelumab (e.g., the Kabat- defined CDRs, the AbM-defined CDRs, or the Chothia-defined CDRs).
  • the PD-L1 antagonist is an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of avelumab. 4.
  • compositions Comprising CD80 Extracellular Domain Fc Fusion Proteins and/or PD-1/PD-L1 Antagonists
  • methods of administering pharmaceutical compositions comprising CD80 ECD-Fc fusion proteins and/or PD-1/PD-L1 antagonists, e.g. having the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. (See, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed.
  • compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.
  • a pharmaceutical composition comprising a CD80 ECD-Fc fusion protein (e.g.
  • a pharmaceutical composition comprising a PD-1/PD-L1 antagonist e.g., a PD-1 antibody or antigen-binding fragment thereof such as pembrolizumab
  • a pharmaceutical composition comprising a PD-1/PD-L1 antagonist e.g., a PD-1 antibody or antigen-binding fragment thereof such as pembrolizumab
  • a pharmaceutical composition comprising a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a pharmaceutical composition comprising a CD80 ECD-Fc fusion protein e.g.
  • a pharmaceutical composition comprises about 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 630 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 560 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a pharmaceutical composition comprises about 420 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 280 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 210 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 140 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 70 mg of a CD80 ECD-Fc fusion protein (e.g.
  • a pharmaceutical composition comprises about 42 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 21 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 7 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 2.1 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a pharmaceutical composition comprises about 0.7 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 0.21 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises about 0.07 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a pharmaceutical composition is formulated for administration of about 700 mg, about 630 mg, about 560 mg, about 420 mg, about 280 mg, about 210 mg, about 140 mg, about 70 mg, about 42 mg, about 21 mg, about 7 mg, about 2.1 mg, about 0.7 mg, about 0.21 mg, or about 0.07 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • pharmaceutical composition comprising a CD80 ECD-Fc fusion protein (e.g.
  • composition comprising SEQ ID NO:5) (e.g., formulated for administration of about 700 mg, about 630 mg, about 560 mg, about 420 mg, about 280 mg, about 210 mg, about 140 mg, about 70 mg, about 42 mg, about 21 mg, about 7 mg, about 2.1 mg, about 0.7 mg, about 0.21 mg, or about 0.07 mg of the CD80 ECD-Fc fusion protein) is for intravenous administration.
  • pharmaceutical composition comprising a CD80 ECD-Fc fusion protein (e.g.
  • composition comprising SEQ ID NO:5) (e.g., formulated for administration of about 700 mg, about 630 mg, about 560 mg, about 420 mg, about 280 mg, about 210 mg, about 140 mg, about 70 mg, about 42 mg, about 21 mg, about 7 mg, about 2.1 mg, about 0.7 mg, about 0.21 mg, or about 0.07 mg of the CD80 ECD-Fc fusion protein) is for administration about once every three weeks, optionally wherein the administration is intravenous.
  • pharmaceutical composition comprising a CD80 ECD-Fc fusion protein (e.g.
  • composition comprising SEQ ID NO:5) (e.g., formulated for administration of about 700 mg, about 630 mg, about 560 mg, about 420 mg, about 280 mg, about 210 mg, about 140 mg, about 70 mg, about 42 mg, about 21 mg, about 7 mg, about 2.1 mg, about 0.7 mg, about 0.21 mg, or about 0.07 mg of the CD80 ECD-Fc fusion protein) is for administration about once every two weeks, optionally wherein the administration is intravenous.
  • pharmaceutical composition comprising a CD80 ECD-Fc fusion protein (e.g.
  • a pharmaceutical composition comprises 21 to 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a pharmaceutical composition comprises 70 to 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 140 to 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 280 to 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition is formulated for administration of 21 mg to 700 mg, 70 mg to 700 mg, 140 mg to 700 mg, or 280 mg to 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • composition comprising a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5) (e.g., formulated for administration of 21 mg to 700 mg, 70 mg to 700 mg, 140 mg to 700 mg, or 280 mg to 700 mg of the CD80 ECD- Fc fusion protein) is for administration about once every three weeks, optionally wherein the administration is intravenous.
  • pharmaceutical composition comprising a CD80 ECD-Fc fusion protein e.g.
  • composition comprising a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5) (e.g., formulated for administration of 21 mg to 700 mg, 70 mg to 700 mg, 140 mg to 700 mg, or 280 mg to 700 mg of the CD80 ECD- Fc fusion protein) is for administration about once every two weeks, optionally wherein the administration is intravenous.
  • pharmaceutical composition comprising a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5) (e.g., formulated for administration of 21 mg to 700 mg, 70 mg to 700 mg, 140 mg to 700 mg, or 280 mg to 700 mg of the CD80 ECD- Fc fusion protein) is for administration about once every week, optionally wherein the administration is intravenous.
  • a pharmaceutical composition comprises 0.07 to 0.21 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 0.21 to 0.7 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 0.7 to 2.1 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 2.1 to 7 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a pharmaceutical composition comprises 7 to 21 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 21 to 42 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 42 to 70 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 70 to 140 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 140 to 210 mg of a CD80 ECD-Fc fusion protein (e.g.
  • a pharmaceutical composition comprises 210 to 280 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 280 to 420 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 420 to 560 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5). In certain aspects, a pharmaceutical composition comprises 560 to 630 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a pharmaceutical composition comprises 630 to 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • a pharmaceutical composition is formulated for administration of 0.07 mg to 0.21 mg, 0.21 mg to 0.7 mg, 0.7 mg to 2.1 mg, 2.1 mg to 7 mg, 7 mg to 21 mg, 21 mg to 42 mg, 42 mg to 70 mg, 70 mg to 140 mg, 140 mg to 210 mg, 210 mg to 280 mg, 280 mg to 420 mg, 420 mg to 560 mg, 560 mg to 630 mg, or 630 mg to 700 mg of a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5).
  • composition comprising a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5) (e.g., formulated for administration of 0.07 mg to 0.21 mg, 0.21 mg to 0.7 mg, 0.7 mg to 2.1 mg, 2.1 mg to 7 mg, 7 mg to 21 mg, 21 mg to 42 mg, 42 mg to 70 mg, 70 mg to 140 mg, 140 mg to 210 mg, 210 mg to 280 mg, 280 mg to 420 mg, 420 mg to 560 mg, 560 mg to 630 mg, or 630 mg to 700 mg of the CD80 ECD-Fc fusion protein) is for administration about once every three weeks, optionally wherein the administration is intravenous.
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • composition comprising a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5) (e.g., formulated for administration of 0.07 mg to 0.21 mg, 0.21 mg to 0.7 mg, 0.7 mg to 2.1 mg, 2.1 mg to 7 mg, 7 mg to 21 mg, 21 mg to 42 mg, 42 mg to 70 mg, 70 mg to 140 mg, 140 mg to 210 mg, 210 mg to 280 mg, 280 mg to 420 mg, 420 mg to 560 mg, 560 mg to 630 mg, or 630 mg to 700 mg of the CD80 ECD-Fc fusion protein) is for administration about once every two weeks, optionally wherein the administration is intravenous.
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • composition comprising a CD80 ECD-Fc fusion protein (e.g. comprising SEQ ID NO:5) (e.g., formulated for administration of 0.07 mg to 0.21 mg, 0.21 mg to 0.7 mg, 0.7 mg to 2.1 mg, 2.1 mg to 7 mg, 7 mg to 21 mg, 21 mg to 42 mg, 42 mg to 70 mg, 70 mg to 140 mg, 140 mg to 210 mg, 210 mg to 280 mg, 280 mg to 420 mg, 420 mg to 560 mg, 560 mg to 630 mg, or 630 mg to 700 mg of the CD80 ECD-Fc fusion protein) is for administration about once every week, optionally wherein the administration is intravenous.
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a CD80 ECD-Fc fusion protein e.g. comprising SEQ ID NO:5
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 10 to 60 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 15 to 60 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 10 to 40 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 15 to 25 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 20 to 40 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 20 to 30 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 30 to 40 moles of SA per mole CD80 ECD- Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising 10, 15, 20, 25, 30, 35, or 40 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising at least 20 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising at least 25 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising at least 35 moles of SA per mole CD80 ECD-Fc fusion protein.
  • a pharmaceutical composition comprises CD80 ECD-Fc fusion proteins (e.g. comprising SEQ ID NO:5) comprising at least 40 moles of SA per mole CD80 ECD-Fc fusion protein.
  • the pharmaceutical composition can be formulated for intravenous administration.
  • the pharmaceutical composition can be formulated for administration every three weeks, every two weeks, or every week.
  • a pharmaceutical composition comprises 200 mg of an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:12, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:13, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:14, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:16, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:17.
  • the antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:12, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:13, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:14, a VL-C
  • a pharmaceutical composition comprises 200 mg of an anti- PD-1 antibody or antigen-binding fragment thereof, wherein the antibody or antigen- binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:10 and a VL comprising the amino acid sequence of SEQ ID NO:11.
  • a pharmaceutical composition comprises 200 mg of an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:8 and a light chain comprising the amino acid sequence of SEQ ID NO:9.
  • a pharmaceutical composition comprises 200 mg of pembrolizumab or an antigen-binding fragment thereof.
  • the pharmaceutical composition can be formulated for intravenous administration.
  • the pharmaceutical composition can be formulated for administration every three weeks.
  • the pharmaceutical composition can be formulated for intravenous administration every three weeks. 5.
  • Methods and Uses of CD80 Extracellular Domain Fc Fusion Proteins and PD-1/PD- L1 Antagonists [0119] Presented herein are methods for treating a solid tumor in a human subject comprising administering to a subject in need thereof a CD80 ECD-Fc fusion protein.
  • the CD80 ECD-Fc fusion protein can comprise the extracellular domain of human CD80 and the Fc domain of human IgG1 and can be administered e.g., once every three weeks, once every two weeks, or once every week.
  • the fusion protein can be administered in combination with a PD-1/PD-L1 antagonist.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 0.07 mg to about 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5) once every two weeks or once a week.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • a method of treating a solid tumor in a human patient can comprise administering the patient about 700 mg, about 630 mg, about 560 mg, about 420 mg, about 280 mg, about 210 mg, about 140 mg, about 70 mg, about 42 mg, about 21 mg, about 7 mg, about 2.1 mg, about 0.7 mg, about 0.21 mg, or about 0.07 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5) once every two weeks.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • a method of treating a solid tumor in a human patient can comprise administering the patient about 700 mg, about 630 mg, about 560 mg, about 420 mg, about 280 mg, about 210 mg, about 140 mg, about 70 mg, about 42 mg, about 21 mg, about 7 mg, about 2.1 mg, about 0.7 mg, about 0.21 mg, or about 0.07 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5) once a week.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • a method of treating a solid tumor in a human patient can comprise administering the patient 21 mg to 700 mg, 70 mg to 700 mg, 140 mg to 700 mg, or 280 mg to 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5) once every two weeks.
  • a method of treating a solid tumor in a human patient can comprise administering the patient 21 mg to 700 mg, 70 mg to 700 mg, 140 mg to 700 mg, or 280 mg to 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5) once a week.
  • a method of treating a solid tumor in a human patient can comprise administering the patient 0.07 mg to 0.21 mg, 0.21 mg to 0.7 mg, 0.7 mg to 2.1 mg, 2.1 mg to 7 mg, 7 mg to 21 mg, 21 mg to 42 mg, 42 mg to 70 mg, 70 mg to 140 mg, 140 mg to 210 mg, 210 mg to 280 mg, 280 mg to 420 mg, 420 mg to 560 mg, 560 mg to 630 mg, or 630 mg to 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5) once every two weeks.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • a method of treating a solid tumor in a human patient can comprise administering the patient 0.07 mg to 0.21 mg, 0.21 mg to 0.7 mg, 0.7 mg to 2.1 mg, 2.1 mg to 7 mg, 7 mg to 21 mg, 21 mg to 42 mg, 42 mg to 70 mg, 70 mg to 140 mg, 140 mg to 210 mg, 210 mg to 280 mg, 280 mg to 420 mg, 420 mg to 560 mg, 560 mg to 630 mg, or 630 mg to 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5) once a week.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 630 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 560 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 420 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 280 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 210 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 140 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD-1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) 70 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three week and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks, and the PD-1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 42 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 2.1 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three wee and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks, and the PD-1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 0.7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 0.21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient (i) about 0.07 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), and (ii) PD-1/PD-L1 antagonist (e.g., about 200 mg pembrolizumab) wherein (i) and (ii) are administered concurrently or sequentially.
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5
  • PD-1/PD-L1 antagonist e.g., about 200 mg pembrolizumab
  • the fusion protein and the antibody are both administered once every three weeks.
  • the CD80 ECD fusion protein is administered once a week, once every two weeks, or once every three weeks
  • the PD- 1/PD-L1 antagonist is administered once every three weeks.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 21 mg to about 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 70 mg to about 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 140 mg to about 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 280 mg to about 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • the CD80 ECD fusion protein can be administered in combination with a PD-1/PD-L1 antagonist (e.g., pembrolizumab, optionally wherein 200 mg of pembrolizumab is administered once every three weeks).
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 0.07 mg to about 0.21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 0.21 mg to about 0.7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 0.7 mg to about 2.1 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 2.1 mg to about 7 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 7 mg to about 21 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 21 mg to about 42 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 42 mg to about 70 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 70 mg to about 140 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 140 mg to about 210 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 210 mg to about 280 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 280 mg to about 420 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 420 mg to about 560 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 560 mg to about 630 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • a method of treating a solid tumor in a human patient comprises administering to the patient about 630 mg to about 700 mg of a CD80 ECD fusion protein (e.g., comprising the amino acid sequence set forth in SEQ ID NO:5), e.g., once every three weeks, two weeks, or one week.
  • the CD80 ECD fusion protein can be administered in combination with a PD-1/PD-L1 antagonist (e.g., pembrolizumab, optionally wherein 200 mg of pembrolizumab is administered once every three weeks).
  • a CD80 ECD fusion protein e.g., comprising the amino acid sequence set forth in SEQ ID NO:5 can be administered intravenously.
  • the solid tumor can be, for example, an advanced solid tumor. In certain instances, the solid tumor is not a primary central nervous system tumor. [0145] In certain instances, the solid tumor is a lung cancer. [0146] In certain instances, the solid tumor is a colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, endometrial cancer, or sarcoma.
  • the patient to be treated according to the methods provided herein may have received prior therapy with at least one PD-1/PD-L1 antagonist selected from a PD-1 antagonist and a PD-L1 antagonist.
  • the PD-1/PD-L1 antagonist can be, for example, nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.
  • the PD-1/ PD-L1 antagonist may have been administered in an advanced or metastatic setting. In other instances, the patient to be treated according to the methods provided herein has not received prior therapy with a PD-1/PD-L1 antagonist.
  • the patient to be treated according to the methods provided herein may have received prior therapy with an anti-angiogenic agent.
  • the anti-angiogenic agent can be, for example, sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab.
  • the anti-angiogenic agent may have been administered in an advanced or metastatic setting.
  • the patient to be treated according to the methods provided herein for example a patient with a melanoma, may have a BRAF mutation.
  • the patient may have received prior therapy with a BRAF inhibitor.
  • the BRAF inhibitor can be, for example, vemurafenib and dabrafenib.
  • the BRAF inhibitor may have been administered in an advanced or metastatic setting.
  • the tumor to be treated according to the methods provided herein can be recurrent or progressive after a therapy selected from surgery, chemotherapy, radiation therapy, and a combination thereof.
  • the tumor to be treated according to the methods provided herein can be resistant or non-responsive to a PD-1/PD-L1 antagonist, such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.
  • the tumor to be treated according to the methods provided herein can be resistant or non-responsive to an anti-angiogenic agent, such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab.
  • an anti-angiogenic agent such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab.
  • a BRAF inhibitor such as vemurafenib or dabrafenib.
  • the tumor to be treated according to the methods provided herein can be refractory to a PD-1/PD-L1 antagonist, such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.
  • a PD-1/PD-L1 antagonist such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.
  • the tumor to be treated according to the methods provided herein can be refractory to an anti-angiogenic agent, such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab.
  • the tumor to be treated according to the methods provided herein can be refractory to a BRAF inhibitor, such as vemurafenib or dabrafenib.
  • a BRAF inhibitor such as vemurafenib or dabrafenib.
  • the tumor to be treated according to the methods provided herein can be recurrent after treatment with a PD-1/PD-L1 antagonist, such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.
  • the tumor to be treated according to the methods provided herein can be recurrent after treatment with an anti-angiogenic agent, such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab.
  • an anti-angiogenic agent such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab.
  • a BRAF inhibitor such as vemurafenib or dabrafenib.
  • the present invention relates to (i) CD80 ECD-Fc fusion protein or pharmaceutical composition comprising the same and (ii) a PD-1/PD-L1 antibody or antigen-binding fragment thereof or pharmaceutical composition comprising the same for use as a medicament for the treatment of a solid tumor, wherein the fusion protein is for administration at 0.7 mg to 700 mg (e.g., 0.07 mg, 0.21 mg, 0.7 mg, 2.1 mg, 7 mg, 21 mg, 42 mg, 70 mg, 140 mg, 210 mg, 280 mg, 420 mg, 560 mg, 630 mg, or 700 mg), e.g., once every three weeks, two weeks, or week, and wherein the antibody is for administration at 200 mg, e.g.
  • the present invention relates to (i) an CD80 ECD-Fc fusion protein or pharmaceutical composition comprising the same and (ii) a PD-1/PD-L1 antibody or antigen-binding fragment thereof, for use in a method for the treatment of a solid tumor wherein 0.7 mg to 700 mg (e.g., 0.07 mg, 0.21 mg, 0.7 mg, 2.1 mg, 7 mg, 21 mg, 42 mg, 70 mg, 140 mg, 210 mg, 280 mg, 420 mg, 560 mg, 630 mg, or 700 mg) of the CD80 ECD-Fc fusion is administered, e.g., once every three weeks, two weeks, or week, and 200 mg of the antibody or antigen-binding fragment thereof is administered, e.g.
  • 0.7 mg to 700 mg e.g., 0.07 mg, 0.21 mg, 0.7 mg, 2.1 mg, 7 mg, 21 mg, 42 mg, 70 mg, 140 mg, 210 mg, 280 mg, 420 mg, 560 mg, 630 mg, or
  • CD80-Fc A human CD80 ECD IgG1 Fc fusion protein (“CD80-Fc”) was bound to magnetic protein-A beads (Life Technologies) in T-cell proliferation media containing RPMI 1640, 100 IU Penicillin/100 ⁇ g/ml Streptomycin, 2 mM L-Glutamine, 100 nM non-essential amino acids, 55 ⁇ M 2-mercaptoethanol and 10% ultra low-IgG fetal bovine serum. Binding reactions were carried out in 96-well flat-bottom tissue culture plates at a volume of 100 ⁇ l per well with a bead concentration of 3 million beads per ml.
  • CD80-Fc was bound to the beads across a series of concentrations: 10, 1, 0.1 ⁇ g/ml.
  • An additional set of binding reactions was also performed with the addition of 3 ng/ml OKT3-scFv. Proteins were allowed to bind for 1 hour at room temperature on a rocking platform, following which 100 ⁇ l of 20 ⁇ g/ml (final concentration 10 ⁇ g/ml) IgG1 Free-Fc (FPT) was added to each well and allowed to bind for an additional hour in order to block any unoccupied Protein-A binding sites on the beads. The fully loaded and blocked beads were then washed 3 times with PBS using a magnetic 96-well plate stand in order to remove unbound proteins.
  • PBMCs Human peripheral blood mononuclear cells
  • T-cells were seeded at a density of 1 million cells/ml in T225 tissue culture flasks in proliferation media (above) supplemented with 8 ng/ml IL-2 and Human T-cell Activator Dynabeads ® (Life Tech) 1 bead/cell. Following seeding, cells were fed with fresh IL-2 and continually kept at a concentration of 0.3 million cells/ml by the addition of fresh proliferation media every 2 days. Cells were kept in a 37 ⁇ C water-jacketed incubator maintained at 5% CO2. After 6 days of expansion, the activator-beads were removed using a magnetic tube stand and the cells were resuspended at a concentration of 1 million cells/ml in fresh proliferation media without IL-2.
  • Example 2 Effects of a CD80 ECD-Fc Fusion Molecule on CT26 Tumors In Vivo with Fc Domains with Different Sialic Acid (SA) Content
  • SA Sialic Acid
  • Seven-week-old female BALB/c mice were purchased from Charles River Laboratories (Hollister, CA) and were acclimated for one week before the study was initiated.
  • the murine colorectal carcinoma cell line CT26 was implanted subcutaneously over the right flank of the mice at 1.0x10 6 cells/200 ⁇ l/mouse. Prior to inoculation, the cells were cultured for no more than three passages in RPMI 1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamine. Cells were grown at 37°C in a humidified atmosphere with 5% CO 2 . Upon reaching 80-85% confluence, cells were harvested and resuspended in a 1:1 mixture of serum-free RPMI 1640 and Matrigel ® at 5 x10 6 cells per milliliter. [0165] Mice were monitored for tumor growth twice weekly following cell implantation.
  • FBS heat-inactivated Fetal Bovine Serum
  • tumor volume (mm 3 ) (width (mm) x length (mm)) 2 /2.
  • the mean tumor volume for all animals enrolled was 94 mm 3 .
  • the first group was injected with 200 ⁇ l of PBS (control) intravenously (i.v.) into the tail vein.
  • the second group was injected with CD80 ECD-Fc at 20 mol SA/mol protein (lot E) i.v. dosed at 0.3 mg/kg.
  • the third group was injected with CD80 ECD-Fc at 20 mol SA/mol protein (lot E) i.v. dosed at 0.6 mg/kg.
  • the fourth group was injected with CD80 ECD-Fc at 15 mol SA/mol protein (lot D) i.v. dosed at 0.3 mg/kg.
  • the fifth group was injected with CD80 ECD-Fc at 15 mol SA/mol protein (lot D) i.v. dosed at 0.6 mg/kg.
  • the sixth group was injected with CD80 ECD-Fc at 5 mol SA/mol protein (lot A) i.v. dosed at 0.3 mg/kg.
  • the seventh group was injected with CD80 ECD-Fc at 5 mol SA/mol protein (lot A) i.v.
  • CT26 tumor model [0169] Seven-week-old female BALB/c mice were purchased from Charles River Laboratories (Hollister, CA) and were acclimated for one week before the study was initiated. The murine colorectal carcinoma cell line CT26 was implanted subcutaneously over the right flank of the mice at 1.0x10 6 cells/200 ⁇ l/mouse.
  • the fourth group was injected with anti-CTLA4 antibody clone 9D9 (IgG2b) i.p. dosed at 10 mg/kg. Tumors were measured on days 10, 13, 17, 19, 21, and 24. [0171] At day 21 (when all the controls were still in the study), treatment with murine CD80 ECD-Fc at 20 mol/mol SA dosed at 0.3 mg/kg resulted in 90% inhibition of tumor growth compared to the control (p ⁇ 0.001). Treatment with anti-CTLA4 antibody at 10 mg/kg resulted in 75% inhibition of tumor growth compared to the control (P ⁇ 0.001). By comparison, treatment with anti-CTLA4 antibody at 1.5 mg/kg only resulted in 53% inhibition of tumor growth (P ⁇ 0.001) (Fig 3).
  • MC38 tumor model [0173] Seven-week-old female C57Bl/6 mice were purchased from Charles River Laboratories (Hollister, CA) and were acclimated for one week before the study was initiated.
  • the murine colorectal carcinoma cell line MC38 was implanted subcutaneously over the right flank of the mice at 0.5x10 6 cells/100 ⁇ l/mouse. Prior to inoculation, the cells were cultured for no more than three passages in RPMI 1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamine. Cells were grown at 37°C in a humidified atmosphere with 5% CO2.
  • FBS heat-inactivated Fetal Bovine Serum
  • the second group was injected with murine CD80 ECD-Fc 20 mol/mol SA i.v. dosed at 3 mg/kg.
  • the third group was injected with anti-CTLA4 antibody clone 9D9 (IgG2b) i.p. dosed at 1.5 mg/kg.
  • the fourth group was injected with anti-CTLA4 antibody clone 9D9 (IgG2b) i.p. dosed at 10 mg/kg. Tumors were measured on days 11, 14, 17, and 19.
  • the murine melanoma cell line B16-F10 was implanted subcutaneously over the right flank of the mice at 0.5x10 6 cells/100 ⁇ l/mouse. Prior to inoculation, the cells were cultured for no more than three passages in DMEM medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamine. Cells were grown at 37°C in a humidified atmosphere with 5% CO2. Upon reaching 80-85% confluence, cells were harvested and resuspended in a 1:1 mixture of serum-free DMEM and matrigel. [0178] Mice were monitored twice weekly following cell implantation for tumor growth.
  • FBS Fetal Bovine Serum
  • tumor volume (mm 3 ) (width (mm) x length (mm)) 2 /2.
  • the mean tumor volume for all animals enrolled was 70 mm 3 .
  • Mice were dosed 3 times: on day 3, 6 and 10.
  • the first group was injected with mouse IgG2b (mIgG2b) dosed i.p. at 10 mg/kg (control).
  • the second group was injected with murine CD80 ECD-Fc 20 mol/mol SA i.v. dosed at 3 mg/kg.
  • the third group was injected with anti-CTLA4 antibody clone 9D9 (IgG2b) i.p. dosed at 1.5 mg/kg.
  • the fourth group was injected with anti-CTLA4 antibody clone 9D9 (IgG2b) i.p. dosed at 10 mg/kg.
  • Tumors were measured on days 10, 13, 15, 16, 17.
  • treatment with murine CD80 ECD-Fc at 20 mol/mol SA dosed at 3 mg/kg resulted in 41% inhibition of tumor growth compared to the control (P ⁇ 0.001).
  • Treatment with anti-CTLA4 antibody at 10 mg/kg or 1.5 mg/kg did not significantly affect tumor growth compared to the control (Fig.5).
  • SPR surface plasmon resonance
  • ELISA enzyme-linked immunosorbent assay
  • flow cytometry flow cytometry.
  • hCD80ECD:hIgG1Fc has the highest affinity for CTLA-4 (1.8 nM), moderate affinity for PD-L1 (183 nM), and low affinity for CD28 (> 1 ⁇ M).
  • the low affinity of hCD80ECD:hIgG1Fc for CD28 is consistent with literature reports.
  • the maximum observed serum concentration (Cmax) of hCD80ECD:hIgG1Fc increased more than dose proportionally from 0.03 mg/kg to 0.9 mg/kg and dose proportionally from 0.9 mg/kg to 3 mg/kg.
  • the area under serum concentration (AUC)-time curve from day 0 to day 4 increased in a dose-proportional manner from 0.03 mg/kg to 3 mg/kg with estimated clearance of 18.0 to 26.3 mL/day/kg and terminal half-life of 1-2 days.
  • both C max and the AUC- time curve from day 0 to day 7 increased approximately in proportion with dose level in the dose range from 1 mg/kg to 100 mg/kg following the first and fourth doses.
  • the estimated terminal half-life was 4 to 6 days. Following 4-weekly dose administration, there was little to no accumulation.
  • Anti-drug antibodies (ADA) were present in the majority of rats (11/16 and 23/24 for the PK study and the GLP toxicology study, respectively). Seven out of 12 and 2 out of 30 cynomolgus monkeys treated with hCD80ECD:hIgG1Fc from the pilot toxicology study and the GLP toxicology study, respectively, were ADA-positive.
  • hCD80ECD:hIgG1Fc has linear clearance for the dose range from 0.03 mg/kg to 3 mg/kg in mice and from 1 mg/kg to 100 mg/kg in rats and cynomolgus monkeys.
  • HCD80ECD:hIgG1Fc has faster clearance and shorter half-life than a typical monoclonal antibody (mAb) in animals.
  • mAb monoclonal antibody
  • Example 6 Toxicology [0189] Toxicology studies were also performed with hCD80ECD:hIgG1Fc. These studies include a pilot repeat-dose toxicity study in cynomolgus monkeys and Investigational New Drug (IND) application-enabling GLP repeat-dose toxicity studies in rats and cynomolgus monkeys. [0190] In the repeat-dose GLP toxicology studies in rats, hCD80ECD:hIgG1Fc was administered at dose levels of 0 (vehicle), 1, 10, or 100 mg/kg/dose for 4 weekly doses. Reversibility of toxicity was evaluated during a 7-week recovery period following the final administration.
  • HCD80ECD:hIgG1Fc was clinically well tolerated in rats up to 100 mg/kg.
  • changes in hematologic parameters were observed, including increases in neutrophils, lymphocytes, and monocytes; a slight decrease in red blood cells (RBCs) and an increase in reticulocytes.
  • Changes in clinical chemistry parameters were mostly seen at 100 mg/kg, including a decrease in triglycerides, an increase in alanine aminotransferase (ALT) and alkaline phosphatase (ALP), a decrease in albumin and an increase in globulins, with an associated decrease in the albumin/globulin ratio.
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • Mononuclear cell inflammation was seen in the stomach, intestine, pancreas, salivary gland, and Harderian gland and was primarily observed at 100 mg/kg with only rare and minimal findings at 10 mg/kg.
  • Increased lymphoid cellularity was observed in lymph nodes, spleen, and gut-associated lymphoid tissue (GALT) and was also primarily observed at 100 mg/kg, with lower frequency and less extensive changes observed at 10 mg/kg.
  • GALT gut-associated lymphoid tissue
  • NOAEL no-observed-adverse-effect level
  • cynomolgus monkeys received 4 weekly IV doses of 0 (vehicle), 1, 10, and 50 mg/kg of hCD80ECD:hIgG1Fc. All dose levels were well tolerated by cynomolgus monkeys. Immunophenotyping analysis showed hCD80ECD:hIgG1Fc-related dose-dependent expansion and proliferation of central memory T-cells in the 10 mg/kg and 50 mg/kg dose groups, but not in the 1 mg/kg group.
  • hCD80ECD:hIgG1Fc protein was administered at dose levels of 0 (vehicle), 1, 10, or 100 mg/kg/dose for 4 weekly doses. Reversibility of toxicity was evaluated during a 6-week recovery period following administration of the last dose.
  • HCD80ECD:hIgG1Fc was well tolerated and no clinical or pathological changes were identified at 1 mg/kg when given as 4 weekly doses, but hCD80ECD:hIgG1Fc was not tolerated at doses of 10 and 100 mg/kg, necessitating unscheduled sacrifice and necropsy of 6/10 and 4/10 animals, respectively, between study days 14 and 30.
  • the affected animals displayed weight loss and lethargy, had signs consistent with dehydration, and were cold to the touch. Some monkeys had sporadic diarrhea. Significant body weight loss was observed several days prior to euthanasia.
  • Affected animals showed significant electrolyte imbalance, including hyponatremia, blood urea nitrogen (BUN) and creatinine elevation, and signs of acute phase reaction (increased fibrinogen, increased globulin, increased C-reactive protein [CRP], and decreased albumin). Aldosterone and cortisol level were increased and adrenocorticotropic hormone (ACTH) decreased. Hematologic analysis showed a severe reduction of reticulocytes in 5 animals. No coagulation changes were observed.
  • Serum cytokine measurements (IL-1 ⁇ , IL-2, IL-4, IL-6, IL-8, IL-10, IFN- ⁇ , TNF- ⁇ , and granulocyte- macrophage colony-stimulating factor [GM-CSF]) on the day of unscheduled euthanasia showed signs of acute stress responses (TNF- ⁇ and IL8 increases), but the pattern of affected cytokines as well as the magnitude of changes did not indicate an acute cytokine release syndrome (CRS), i.e., no increase in IL2 or IL6.
  • CRS acute cytokine release syndrome
  • Findings of uncertain relationship to hCD80ECD:hIgG1Fc included an increased incidence of tubular dilatation with tubular casts and mineralization in the kidney, and an increased incidence of adrenal hypertrophy (zona fasciculata) in the adrenal.
  • zona fasciculata adrenal hypertrophy
  • HCD80ECD:hIgG1Fc-related changes in clinical chemistry parameters in the 10 mg/kg and 100 mg/kg group included a mild reduction in albumin and a mild increase in globulin at 10 mg/kg and 100 mg/kg. These changes were accompanied by increased fibrinogen, suggestive of an acute phase response. These changes returned to baseline at the end of the recovery period. These changes in clinical chemistry were not observed in the animals that survived to scheduled necropsy. No signs indicative of CRS, such as fever or cytokine increases consistent with CRS events, were observed. [0198] Ophthalmic examination and cardiac evaluation did not show any hCD80ECD:hIgG1Fc-related changes at any dose level.
  • histopathological mucosal erosion and crypt dilatation were seen in the large intestine of animals given 100 mg/kg with sporadic findings in animals given 10 mg/kg. Also, at the scheduled necropsy, increased lymphoid cellularity was observed in the lymph nodes, whereas decreased lymphoid cellularity was observed in the spleen and thymus. [0199] Overall, the histopathological changes were not of a magnitude that would explain the observed moribundity at doses of ⁇ 10 mg/kg. The changes observed in the intestine were minimal to mild, and the diarrhea was sporadic among the affected animals.
  • hCD80ECD:hIgG1Fc was clinically well tolerated in rat, and the NOAEL in rats is considered 10 mg/kg for 4-weekly doses.
  • doses of 10 mg/kg and 100 mg/kg were not tolerated.
  • Some monkeys at the 10 mg/kg dose had sporadic diarrhea, dehydration, lethargy, and were cold to the touch.
  • Intravenous hydration only temporarily improved the symptoms. Diffuse lymphocytic and monocytic infiltrates were observed in a variety of organs, however, the mechanism of this toxicity is undetermined.
  • the starting dose of 0.07 mg (0.001 mg/kg for a 70 kg human) has been calculated based on the minimum anticipated biologic effect level (MABEL) approach (see Example 7 below) and is approximately 1000-fold below the NOAEL.
  • Significant anti-tumor activity is evident even at doses as low as 0.1 mg/kg in the CT26 tumor model, which is approximately 10-fold below the NOAEL in both rats and monkeys. Therefore, a potential therapeutic window for hCD80ECD:hIgG1Fc exists.
  • Example 7 Selection of Initial Dose for Human Patients [0201] A conservative starting dose based on the MABEL approach, close patient monitoring, staggered enrollment, and cautious dose escalation was designed to limit the risk to patients. [0202] The MABEL approach was used because hCD80ECD:hIgG1Fc functions through two key T-cell regulators or modulators, including co-stimulation of CD28 on T-cells after T- cell receptor engagement, and blocking of CTLA-4 from competing for endogenous CD80. For hCD80ECD:hIgG1Fc, assessments of receptor occupancy (RO) and pharmacological activity (PA) through both CTLA-4 and CD28 were considered.
  • RO receptor occupancy
  • PA pharmacological activity
  • hCD80ECD:hIgG1Fc is predicted to be less than 10 days, preclinical evidence suggests that the total exposure, not Ctrough, may be an important driver of efficacy.
  • the starting dose of 0.07 mg is predicted to attain a nominal ( ⁇ 1%) PA for CD28 using the binding assay of Chinese hamster ovary (CHO) cells overexpressing CD28.
  • the dose escalation cohorts, along with the predicted PA for CD28 and CTLA-4 at each dose level at Cmax, is summarized below (Table 2).
  • Table 2 The dose escalation cohorts, along with the predicted PA for CD28 and CTLA-4 at each dose level at Cmax.
  • hCD80ECD:hIgG1Fc is projected to achieve 99% PA for CTLA-4 at C max for doses ⁇ 7 mg.
  • ipilimumab an anti-CTLA4 antibody, was projected to achieve 99% RO for CTLA- 4 at the clinically approved dose of 3 mg/kg.
  • Table 2 hCD80ECD:hIgG1Fc Dose Selection *PA estimation based on IC 50 of 16,000 ng/ml from cell binding assay using CD28 overexpressed CHO cell lines. **PA estimation based on EC50 of 34 ng/mL from hCD80ECD:hIgG1Fc CTLA-4 binding ELISA [0205] Thus, the selected human doses take into account RO and PA through both CD28 and CTLA-4.
  • Phase 1a Dose Escalation and Exploration Study A phase 1a open-label multicenter study is conducted in up to 78 patients with advanced solid tumors using hCD80ECD:hIgG1Fc. Some patients may be enrolled at one or more dose levels. The patients in this study have advanced solid tumors, except central nervous system tumors. The patients are refractory to all standard therapies for their malignancy or are patients for whom standard therapies would not be appropriate.
  • Phase 1a includes a Dose Escalation phase and a Dose Exploration phase.
  • the Phase 1a study schema is provided in Fig.6.
  • hCD80ECD:hIgG1Fc is administered as a 60-minute intravenous (IV) infusion every three weeks (Q3W) on Day 1 of each 21-day cycle.
  • HCD80ECD:hIgG1Fc is administered as a flat dose.
  • the Phase 1a Dose Escalation includes an initial accelerated titration design followed by a standard 3+3 dose escalation design until the recommended dose (RD) for Phase 1b is determined. Up to 48 patients participate in the Dose Escalation phase.
  • Doses from 0.07 mg to 70 mg are administered per the cohorts outlined in Table 3 below, and patients' second doses are at least 21 days after their first doses. [0209] As immuno-oncology agents are associated with delayed immune-mediated toxicities, toxicities observed both during and beyond the 21-day dose-limiting toxicity (DLT) evaluation period are evaluated. Table 3: Dose Levels for Accelerated Titration Design and 3+3 Design [0210] During Phase 1a Dose Escalation, the Dose-Limiting Toxicity (DLT) evaluation begins on the first day of treatment upon start of infusion and continues for 21 days.
  • DLT Dose-Limiting Toxicity
  • a DLT is defined as any of the following as related hCD80ECD:hIgG1Fc: (i) Absolute Neutrophil Count (ANC) is less than 1.0 ⁇ 10 9 per L for more than 5 days or Grade 3 febrile neutropenia (e.g., ANC less than 1.0 ⁇ 10 9 per L with a single temperature of more than 38.3°C or fever more than 38°C for more than 1 hour); (ii) platelets are less than 25 ⁇ 10 9 per L or platelets are less than 50 ⁇ 10 9 per L with clinically significant hemorrhage; (iii) aspartate aminotransferase/alanine transaminase (AST/ALT) is more than 3 times the upper limit of normal (ULN), and concurrent total bilirubin is more than twice ULN not related to liver involvement with cancer; (iv) Grade 3 or higher non- hematologic toxicity (except Grade 3 fatigue lasting less than 7 days; Grade 3 nausea and Grade 3-4 vomiting and diarrhea lasting less than
  • An accelerated titration design enrolling at least 1 patient at each dose level is carried out for dose levels 0.07, 0.21, 0.7 and 2.1 mg. Dose escalation to the next dose level proceeds after at least 1 patient completes the 21-day DLT evaluation interval. If a single patient experiences a DLT during the 21-day evaluation interval, standard 3+3 dose escalation criteria applies for that cohort as well as all subsequent dosing cohorts. If at least 2 patients experience moderate adverse events (AE) (at any accelerated titration dose level), standard 3+3 dose escalation criteria will apply for the highest dose level at which a moderate AE was experienced, with enrollment of additional patients. All subsequent dosing cohorts will then follow the standard 3+3 dose escalation criteria.
  • AE adverse events
  • Moderate AEs are defined as ⁇ Grade 2 AEs as related to hCD80ECD:hIgG1Fc.
  • Grade 2 laboratory values are not considered as moderate AEs for this purpose unless accompanied by clinical sequelae.
  • Intra-patient dose escalation will be permitted in patients enrolled at dose levels below 7.0 mg provided: (i) the patient did not experience a DLT; (ii) all other AEs have recovered to Grade 1 or lower prior to dose escalation; (iii) the patient may only dose escalate by a maximum of 1 dose level every 21 days and only after that dose level has cleared DLT review; and (iv) the patient cannot dose escalate beyond the 7.0 mg dose level.
  • the algorithm outlined in Table 4 below is used for all standard 3+3 dose escalations. Table 4: Phase 1a Algorithm for 3+3 Dose Escalation Decisions
  • the maximum tolerated dose (MTD) and/or recommended dose (RD) of hCD80ECD:hIgG1Fc for Phase 1a is identified based on an evaluation of the overall safety, tolerability, pharmacodynamics, pharmacokinetics, and preliminary efficacy.
  • the MTD will be a dose level where no more than 1/6 patients report a DLT.
  • the RD will be identified based on an evaluation of all available safety, tolerability, pharmacokinetic, and pharmacodynamics data.
  • the RD will consider toxicities observed both during and beyond the DLT evaluation period as well as dose reductions and discontinuations due to toxicity that do not meet the DLT criteria.
  • the RD therefore, may or may not be the same as the identified MTD.
  • the Phase 1a Dose Exploration cohort enrolls up to 30 patients in total who may be enrolled at one or more dose levels to further evaluate safety, pharmacokinetics, pharmacodynamics, and clinical activity. Toxicities observed in these patients will contribute to the overall assessments of safety and tolerability, and may inform selection of the RD. Clinical activity may be evaluated in specific tumor types based on safety, pharmacokinetic, pharmacodynamic, and efficacy data.
  • Cytokine levels including circulating IL-6, TNF, and IFN ⁇ levels are monitored.
  • SUBJECTS [0217] A total of up to 78 patients in Phase 1a are identified based on the following inclusion and exclusion criteria.
  • Patients in Phase 1a meet all of the following inclusion criteria: • Patients must be 18 years of age or older • Histologically confirmed solid tumors (except primary central nervous system tumors); • Disease that is unresectable, locally advanced, or metastatic and has progressed following all standard treatments (i.e., refractory) or is not appropriate for standard treatments; • At least one measurable lesion at baseline according to RECIST v1.1; tumor sites situated in a previously irradiated area, or in an area subjected to other loco-reginal therapy, are not considered measurable unless there has been demonstrated progression in the lesion; [0219] Patients in Phase 1a are excluded from the study if any of the following criteria apply: • Treatment with any anti-cancer therapy or participation in another investigational drug or biologics trial within 28 days or ⁇ 5 half-lives (whichever is shorter) prior to first dose of study treatment administration or while on this study; • For patients participating in Phase 1a dose escalation and exploration cohorts: Prior treatment with a CTLA-4 antagonist
  • AEs defined as dose-limiting toxicities
  • clinical laboratory abnormalities defined as dose-limiting toxicities
  • overall assessment of pharmacokinetics and pharmacodynamics are evaluated to determine the recommended dose of hCD80ECD:hIgG1Fc.
  • Pharmacokinetic parameters (AUC, Cmax, Ctrough, CL, t1/2, vss (volume of distribution at a steady state)) in patients with advanced solid tumors are determined from serum concentration-time data of hCD80ECD:hIgG1Fc using a non-compartmental analysis. Other parameters, such as dose proportionality, accumulation ratio, and attainment of steady state, will also be calculated if the data are available.
  • Serum concentrations of hCD80ECD:hIgG1Fc are determined using the enzyme-linked immunosorbent assay (ELISA) method.
  • ELISA enzyme-linked immunosorbent assay
  • Tumor assessments include a clinical examination and imaging (e.g., computed tomography (CT) scans with appropriate slice thickness per RECIST v1.1 or magnetic resonance imaging (MRI)). Tumors are assessed at screening, every 6 weeks from the first dose for 24 weeks, then every 12 weeks thereafter to show inhibition of tumor growth and tumor regression (e.g., complete tumor regression). Once an initial CR or PR is noted, confirmatory scans must be performed 4 to 6 weeks later. A lack of significant increase in circulating IL-6, TNF, and IFN ⁇ indicates that hCD80ECD:hIgG1Fc does not cause a cytokine storm. [0224] The objective response rate (ORR) is also determined as a measure of efficacy.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • the ORR is defined as the total number of patients with confirmed responses (either complete response (CR) or partial response (PR) per RECIST v.1.1) divided by the total number of patients who are evaluable for a response.
  • CR complete response
  • PR partial response
  • T cell proliferation was observed in patients that received 42 mg or more of CD80ECD:hIgG1Fc.
  • Phase 1b Dose Expansion A Phase 1b open-label multicenter study is conducted using hCD80ECD:hIgG1Fc in up to 180 patients with advanced solid tumors. (A) STUDY DESIGN [0228] Phase 1b is the dose expansion portion of the study. The Phase 1b study schema is provided in Fig.6.
  • Phase 1b includes tumor-specific cohorts of up to 30 patients each as shown in Table 5. Patients with renal cell carcinoma or melanoma who have failed prior anti-PD(L)1 therapy are enrolled. Additional tumor types for the remaining four Phase 1b cohorts will be determined based on safety, translational, and safety information from other immunotherapies and changes to prescribing information for approved immunotherapies.
  • HCD80ECD:hIgG1Fc is administered as a 60-minute intravenous (IV) dose every three weeks (Q3W) on Day 1 of each 21-day cycle.
  • HCD80ECD:hIgG1Fc is administered as a flat dose.
  • SUBJECTS Up to 30 patients are enrolled into each specific Phase 1b cohort.
  • Phase 1b Patients in Phase 1b meet all of the following inclusion criteria: • All inclusion criteria for Phase 1a; • For Cohort 1b1 – renal cell carcinoma o Histologically or cytologically confirmed advanced or metastatic renal cell carcinoma with a clear-cell component; o Patients must have received at least one prior anti-angiogenic therapy regimen (e.g., sunitinib, sorafenib, pazopanib, axitinib, tivozanib, or bevacizumab) in the advanced or metastatic setting; and o Patients must have received at least one anti-PD(L)1 therapy (e.g., nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab) in the advanced or metastatic setting.
  • anti-PD(L)1 therapy e.g., nivolumab, pembrolizumab, atezolizumab, durvalumab, or ave
  • Prior cytokine therapy e.g., IL-2 or IFN- ⁇
  • anti-CTLA4 therapy e.g., ipilimumab
  • anti-PD(L)1 therapy e.g., nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab
  • Prior cytokine therapy e.g., IL-2 or IFN- ⁇
  • anti-CTLA4 therapy e.g., ipilimumab
  • Prior cytokine therapy e.g., IL-2 or IFN- ⁇
  • anti-CTLA4 therapy e.g., ipilimumab
  • prior BRAF inhibitor therapy e.g., vemurafenib or dabrafenib
  • C RESULTS
  • AEs AEs, serious AEs, clinical laboratory abnormalities, and electrocardiogram (ECG) abnormalities are evaluated to show that hCD80ECD:hIgG1Fc is safe and tolerable in patients with advanced solid tumors.
  • ECG electrocardiogram
  • the incidence of AEs defined as dose-limiting toxicities, clinical laboratory abnormalities defined as dose-limiting toxicities, and overall assessment of pharmacokinetics and pharmacodynamics are evaluated to determine the recommended dose of hCD80ECD:hIgG1Fc.
  • Pharmacokinetic parameters (AUC, Cmax, Ctrough, CL, t1/2, vss (volume of distribution at a steady state)) in patients with advanced solid tumors are determined from hCD80ECD:hIgG1Fc serum concentration-time data using a non-compartmental analysis. Other parameters, such as dose proportionality, accumulation ratio, attainment of steady state, will also be calculated if the data are available. Serum concentrations of hCD80ECD:hIgG1Fc are determined using the enzyme-linked immunosorbent assay (ELISA) method.
  • ELISA enzyme-linked immunosorbent assay
  • hCD80ECD:hIgG1Fc The impact of immunogenicity (i.e., anti-drug antibody immune responses to hCD80ECD:hIgG1Fc) in patients with advanced solid tumors on exposure to hCD80ECD:hIgG1Fc is assessed by measuring total antibodies against hCD80ECD:hIgG1Fc from all patients.
  • Tumor assessments include a clinical examination and imaging (e.g., computed tomography (CT) scans with appropriate slice thickness per RECIST v1.1 or magnetic resonance imaging (MRI)).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • the objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), disease control rate (DCR), and overall survival (OS) are also determined as a measure of efficacy.
  • the ORR is defined as the total number of patients with confirmed responses (either complete response (CR) or partial response (PR) per RECIST v.1.1) divided by the total number of patients who are evaluable for a response.
  • the DOR is defined as the time from first response (CR or PR per RECIST v1.1) that is subsequently confirmed until the onset of progressive disease or death from any cause, whichever comes first.
  • PFS is defined as the time from the patient’s first dose to the first observation of disease progression or death due to any cause, whichever comes first.
  • DCR is defined as the total number of patients with confirmed responses of either CR, PR, or stable disease as per RECIST v1.1 divided by the total number of patients who are evaluable for a response.
  • OS is defined as the time from the first dose of hCD80ECD:hIgG1Fc until death from any cause.
  • HCD80ECD:hIgG1Fc was administered to human patients per the protocols described in Examples 8 and 9. The characteristics of the treated patients are summarized in Table 6.
  • Table 6 Characteristics of Patients Treated with HCD80ECD:hIgG1Fc ⁇ 1 patient in dose-expansion [0240] In these patients, no dose limiting toxicities or adverse events of grade 4 or higher were observed. There were five serious adverse events (including deep vein thrombosis, bile duct obstruction, new CNS lesion; recurrent pleural effusion, and herpes zoster), all due to underlying disease. Anti-drug antibodies were observed in 2 out 15 patients.
  • Stable disease occurred as follows: 0/1 patient receiving 0.7 mg, 1/1 patient receiving 0.21 mg, 1/2 patients receiving 0.7 mg, 1/1 patient receiving 2.1 mg, 1/4 patients receiving 7 mg, and 0/3 patients receiving 21 mg. Conversely, progressive disease occurred as follows: 1/1 patient receiving 0.7 mg, 0/1 patient receiving 0.21 mg, 1/2 patients receiving 0.7 mg, 0/1 patient receiving 2.1 mg, 3/4 patients receiving 7 mg, and 3/3 patients receiving 21 mg.
  • the percentages of CTLA-4 receptor occupancy associated with the Cmax and Ctrough values observed in patients receiving 0.07 mg to 21 mg were calculated based on the binding affinity of hCD80ECD:hIgG1Fc for CTLA-4 (using surface plasmon resonance at 1.8 nM).
  • Cmax and Ctrough values are shown in italics with underline. Other Cmax and Ctrough values are projected values based on pharmacokinetic dates from 3 patients at 21 mg doses (assuming linear clearance) [0244] In order to achieve a desired receptor occupancy of about 60% to about 98.5%, doses of 140 -700 mg were selected.
  • Example 11 Gene Expression Analysis of Granzyme B and Interferon Gamma in Tumor-Bearing and Na ⁇ ve BALB/c Mice Treated with Murine CD80 ECD-Fc
  • Immuno-competent BALB/c mice were inoculated with CT26, a murine colorectal carcinoma, and treatment with murine CD80 ECD-Fc was administered IV when tumors reached approximately 100 mm 3 .
  • Murine CD80 ECD-Fc is a mouse surrogate fusion protein comprising the extracellular domain (ECD) of murine CD80 linked to the Fc domain of mouse IgG2a wild type (mCD80-Fc).
  • Murine CD80 ECD-Fc was evaluated at four dose levels: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, and 0.9 mg/kg.
  • non-tumor-bearing BALB/c mice were administered 0.9 mg/kg, 10 mg/kg, or 50 mg/kg mCD80-Fc.
  • mice were administered 0.9 mg/kg (tumor-bearing) or 50 mg/kg (na ⁇ ve) mIgG2a isotype control. Samples were collected for transcriptomic analysis 11 days post-dose. Tumors were resected and snap-frozen in liquid nitrogen, and blood samples were collected in Qiagen RNAprotect animal blood tubes (100 ⁇ l).
  • Gzmb Granzyme B
  • mCD80-Fc treatment did not impact Gzmb expression in non-tumor-bearing animals except at as the highest dose level tested, 50 mg/kg.
  • Interferon gamma (Ifng) was significantly upregulated at 0.9 mg/kg both in tumor and blood from tumor-bearing mice, with a small trend towards increased expression at 0.3 mg/kg in both compartments.
  • Murine CD80 ECD-Fc treatment only upregulated Ifng expression in blood from na ⁇ ve animals at 50 mg/kg.
  • Example 12 CD80 ECD-Fc Activity in Whole Blood Mixed Lymphocyte Reactions [0247] HCD80ECD:hIgG1Fc was tested in vitro in primary T cells assays using pooled, irradiated PBMC from multiple donors to stimulate individual donor blood T cells (Bromelow et al., Journal of Immunological Methods 247: 1-8 (2000).
  • Alloreactive T cells are found at high frequencies in the blood and react to a variety of peptide:MHC presented on the surface of irradiated PBMC, which also express Fc receptor (FcR) that can bind hCD80ECD:hIgG1Fc and mediate co-stimulation of responding T cells.
  • FcR Fc receptor
  • This format allows the testing of hCD80ECD:hIgG1Fc activity with physiologically-relevant antigen presenting cell (APC) populations, and the use of pooled PBMC helps to reduce donor to donor variability in T cell responses.
  • APC antigen presenting cell
  • PBMCs from each donor were pooled at a final concentration of 1x10 6 cells/mL in RPMI-10 (Roswell Park Memorial Institute 1640 medium supplemented with 2 mM L-glutamine, 25 mM Hepes, 1x Penicillin/ Streptomycin, 2ME and 10% human serum.
  • RPMI-10 Roswell Park Memorial Institute 1640 medium supplemented with 2 mM L-glutamine, 25 mM Hepes, 1x Penicillin/ Streptomycin, 2ME and 10% human serum.
  • Test conditions were prepared at 4x the desired final concentration in media, and the following were combined per well in a 96-well U-bottom tissue culture plate: • 50, 25, or 12.5 ⁇ L of irradiated PBMC (8, 4, and 2x10 5 PBMC, respectively) with RPMI-10 supplemented to 50 ⁇ L total ; • 50 ⁇ l of 1000, 500, or 250 ⁇ g/mL Fc-Hinge control or hCD80ECD:hIgG1Fc for final concentrations of 250, 125, and 62.5 ⁇ g/mL; • 50 ⁇ l of media containing antibody: anti-CD32 at 40 ⁇ g/mL (final 10 ⁇ g/mL), anti- CD28 at 4 ug/mL (final 1 ⁇ g/mL), anti-PD-L1 at 40 ⁇ g/mL (final 10 ⁇ g/mL), or Ipilimumab at 40 ug/mL (final 10 ⁇ g/mL) ;
  • T cell phenotype and function were added to the samples in 100 ⁇ l of FACS buffer and then incubated for 30 minutes at 4oC. The cells were then labeled with FoxP3, intracellular cytokine staining, and Clik-iT EdU.
  • Samples were acquired on a BD LSRFortessa and analyzed using FlowJo, Excel, and Graphpad Prism software. Briefly, singlet events were identified by comparing scatter characteristics, and T cells were identified as Lineage- (CD14-, CD15-, CD19-, and CD56-), CD3+, CD4+ or CD8+ cells.
  • cell-surface markers of activation were also assessed (e.g., CD25, CD95, PD1).
  • Secreted cytokines were measured in assay supernatants by colorimetric ELISA using a commercial kit according to the manufacturer’s instructions. Assay plates were read using an Envision 2103, and the data were analyzed using Excel and Graphpad Prism Software [0253] There were few cytokines produced when stimulated with 2x10 5 or 8x10 5 PBMC in the absence of costimulation. Furthermore, both CD4 and CD8 T cells showed little proliferation or activation-induced upregulation of CD25 without additional signaling.
  • HCD80ECD:hIgG1Fc enhanced IL-2 and IFN ⁇ secretion by T cells, and this effect was dependent upon the number of stimulator cells (Fig.8).
  • HCD80ECD:hIgG1Fc The maximal effect of hCD80ECD:hIgG1Fc was higher than that observed with saturating agonistic anti-CD28. HCD80ECD:hIgG1Fc and also increased the proliferation of CD4 and CD8 T cells and expression of CD25 in a stimulator-dependent manner (Fig.9). However, unlike the cytokine levels, the increases in T cell proliferation were significant when stimulated with 2x10 5 PBMC. CD4 T cell upregulation of CD25 was also observed following stimulation with low and high numbers of PBMC. HCD80ECD:hIgG1Fc did not activate T cells in the absence of TCR stimulation, as evidenced by control samples utilizing whole blood and autologous irradiated PBMC.
  • One assay measured the binding of C1q to human immune cell-bound hCD80ECD:hIgG1Fc using PBMC left unactivated (expressing only CD80 ligands and CD28 and PD-L1) or activated to induce cell surface expression of CTLA-4 in addition to CD28 and PD-L1.
  • PBMC left unactivated expressing only CD80 ligands and CD28 and PD-L1
  • activated to induce cell surface expression of CTLA-4 in addition to CD28 and PD-L1.
  • CD80 ECD-Fc is Active in 200 mm 2 Tumors [0257] The activity of murine CD80 ECD-Fc on CT26 tumors is shown in above in Example 3.
  • mice received saline, 0.3 mg/kg murine CD80 ECD-Fc, 1 mg/kg murine CD80 ECD-Fc, or 3 mg/kg murine CD80 ECD-Fc. As shown in Fig.10, all three doses of murine CD80 ECD-Fc significantly inhibited the growth of CT26 tumors as compared to the saline treatment group.
  • Example 14 CD80 ECD-Fc Activity is Increased in Combination with Anti-PD-1 Antibody [0258] The activity of murine CD80 ECD-Fc in combination with a murine anti-PD-1 antibody on CT26 tumors (a murine colon carcinoma derived from BALB/c mice) was examined. CT26 tumors were subcutaneously injected into immunocompetent BALB/c mice. On Day 7, tumor volumes were measured, and animals were grouped as shown in Table 8. Table 8: Treatment Groups
  • Murine CD80 ECD-Fc (mCD80 ECD-Fc) and msIgG isotype control antibodies were administered as a single dose on Day 10.
  • Anti-PD-1 was administered on Days 10 and 13, for a total of 2 doses. Tumors were measured at least twice weekly until the study concluded on Day 31.
  • the results, shown in Fig.12, demonstrate that mCD80 ECD-Fc significantly reduced tumor volume in the 0.3 mg/kg, 0.1 mg/kg, and the 0.03 mg/kg dosing groups but not in the 0.01 mg/kg dosing group (one-way ANOVA).
  • Anti-PD-1 monotherapy treatment did not result in statistically significant tumor growth reduction.
  • Example 15 Phase 1a Dose Escalation and Exploration Study [0260] A phase 1a/b open-label multicenter study is conducted with advanced lung cancer using hCD80ECD:hIgG1Fc and the anti-PD-1 antibody pembrolizumab.
  • hCD80ECD:hIgG1Fc is administered once every 3 weeks (Q3W) over approximately 60 minutes by intravenous (IV) infusion followed by 200 mg of pembrolizumab administered by IV infusion after the completion of hCD80ECD:hIgG1Fc (by at least 30 minutes). Both the hCD80ECD:hIgG1Fc and the pembrolizumab are administered on Day 1 of the 21-day cycle (Q3W).
  • Dose escalation will start with a hCD80ECD:hIgG1Fc dose that is a minimum of two dose levels lower than the highest dose cleared in hCD80ECD:hIgG1Fc monotherapy. Dose escalation decisions follow the standard 3+3 algorithm described above (see Examples 8-10), and continue up to the dose level established as the maximum tolerated dose (MTD) for hCD80ECD:hIgG1Fc monotherapy.
  • MTD maximum tolerated dose
  • irAEs immune-related adverse events
  • An irAE is defined as a clinically significant adverse event (AE) that is associated with study drug exposure, without a clear alternate cause, and consistent with an immune-mediated mechanism. Based on that background, the first occurrence of the following irAEs will not be considered a DLT because they may occur with immune therapy and are likely to be fully reversible.
  • Grade 3 tumor flare defined as local pain, irritation, or rash localized at sites of known or suspected tumor
  • Grade 3 non-skin immune-related adverse event irAE
  • Transient resolving within 6 hours of onset
  • Grade 3 infusion-related AEs Grade 3 drug-related bronchospasm or anaphylactic or anaphylactoid reactions.
  • Tumor assessments include a clinical examination and imaging (e.g., computed tomography (CT) scans with appropriate slice thickness per RECIST v1.1 or magnetic resonance imaging (MRI)). Tumors are assessed at screening, every 6 weeks from the first dose for 24 weeks, then every 12 weeks thereafter to show inhibition of tumor growth and tumor regression (e.g., complete tumor regression). Once an initial CR or PR is noted, confirmatory scans must be performed 4 to 6 weeks later.
  • the objective response rate (ORR) is also determined as a measure of efficacy.
  • the ORR is defined as the total number of patients with confirmed responses (either complete response (CR) or partial response (PR) per RECIST v.1.1) divided by the total number of patients who are evaluable for a response.
  • No dose limiting toxicities were observed at the 70 mg dose, and an unconfirmed partial response was observed at the 140 mg dose. * * * [0267]
  • the invention is not to be limited in scope by the specific aspects described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

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Abstract

La présente divulgation concerne des méthodes d'administration de protéines de fusion comprenant le domaine extracellulaire du groupe de différenciation 80 (CD80) humain et le domaine de fragment cristallisable (Fc) de l'immunoglobuline G 1 (IgG1) humaine, éventuellement en combinaison avec un antagoniste PD-1/PD-L1, à un sujet en ayant besoin, par exemple, un patient cancéreux.
EP21713285.1A 2020-02-26 2021-02-26 Thérapie basée sur une protéine de fusion fc du domaine extracellulaire cd80 Pending EP4110802A1 (fr)

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